title: Activation of the prostaglandin FP receptor in human granulosa cells.
abstract: Prostaglandin F2 alpha (PGF2 alpha) has regulatory (mainly luteolytic) effects in the ovary but the mechanism of action is not completely understood. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques were used to demonstrate the presence of mRNA encoding the PGF2 alpha receptor (FP receptor) in human granulosa-lutein cells. Specific primers for the amplification of cDNA were designed and yielded a single product of 696 bp corresponding to the FP receptor. The identity of this product was verified by sequencing. Fluprostenol, a selective FP receptor agonist, activated phospholipase C (PLC) and increased intracellular free calcium concentration, confirming the functional activation of the receptor. We have demonstrated by Western blotting that granulosa cells express PLC-beta and PLC-gamma isoforms. The cells responded to pervanadate with increased PLC activity and increased tyrosine phosphorylation, demonstrating a functional PLC-gamma tyrosine kinase pathway. However, fluprostenol did not provoke any detectable tyrosine phosphorylation. Moreover, the effect of fluprostenol was inhibited through protein kinase C stimulation by phorbol 12, 13-dibutyrate, and was not affected when cells were treated with phenylarsine oxide, which blocks tyrosine phosphorylation. These results suggest that the FP receptor activates PLC-beta rather than PLC-gamma isoforms. Fluprostenol-induced activation was pertussis toxin resistant. Granulosa cells express G proteins of the Gq family (resistant to pertussis toxin) and mRNA for both G alpha q and G alpha 1 l has been identified by RT-PCR. In conclusion, human granulosa cells have a functional FP receptor the effects of which are mediated through PLC-beta activation probably via Gq/1 l.
authors.affiliation: Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, UK.
authors.forename: M P
authors.initials: MP
authors.lastname: Carrasco
authors.forename: G
authors.initials: G
authors.lastname: Asbóth
authors.forename: S
authors.initials: S
authors.lastname: Phaneuf
authors.forename: A
authors.initials: A
authors.lastname: López Bernal
mesh_terms: D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D015237:Dinoprost / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism; D006107:Granulosa Cells / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008186:Luteolytic Agents / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011461:Prostaglandins F, Synthetic / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D011982:Receptors, Prostaglandin / Q000235:genetics / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D008186:Luteolytic Agents; D011461:Prostaglandins F, Synthetic; D012333:RNA, Messenger; D011982:Receptors, Prostaglandin; C034671:prostaglandin F2alpha receptor; C006326:fluprostenol; D015237:Dinoprost; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium